Humans and other mammals have extremely limited postnatal regenerative abilities, and these limitations pose a significant challenge to health and quality of life. In contrast to humans, axolotl salamanders regenerate many organs and appendages, such as limbs, with astonishing success. Axolotl limbs are very similar anatomically to human limbs, so they offer an ideal opportunity for discovering regenerative mechanisms that might lead to the development of future therapeutics. In my new laboratory, we are investigating the molecular mechanisms of limb regeneration in axolotls so that we can later apply this knowledge to understanding why humans cannot regenerate limbs. An outstanding question is why highly-regenerative organisms use a structure called a blastema, where internal progenitor cells accumulate, to drive regeneration. Blastema cells are heterogeneous in their lineage and likely their potentials, but very little is known about how these attributes are controlled or even how progenitor cells are cued to become activated and join the blastema. To understand these questions, we have initiated a large RNA-seq based approach, and we are coupling this approach to powerful new tools for examining gene function in these organisms. In our first analysis, we have profiled the transcriptomes of individual cells from two key tissues, at one time point. We also generated a tissue-coded de novo transcriptome to use as a reference for gene assignment and for differential gene expression analysis. The initial individual cells sequenced were fully-formed blastema cells and wound epidermis cells, which overly the blastema and are thought to control key aspects of regeneration. We chose this time point, 23 days post- amputation, as the first sampling point because at this time the blastema population is at its height for numbers of cells but there are not yet any overt signs of differentiation. We have thus far discovered many transcripts that are specifically upregulated in individual cells in these important tissues, and we have performed functional analyses with two of the genes. In this proposal, we aim to use this powerful strategy to identify the gene expression changes that support the transition from intact tissue to activated progenitor cells during the creation of the blastema. We will profile the transcriptomes of more individual cells, but now we will query cells harvested from time points between amputation and the full blastema. In parallel, we will further examine genes uncovered in the first analysis, specifically those that show binary expression patterns and may therefore distinguish subtypes of blastema cells or wound epidermis cells. We will use recently-developed loss-of-function and gain-of-function technologies to interrogate specific genes in vivo, in regenerating limbs. This work is innovative because it takes a completely a priori approach to discovering mechanisms of limb regeneration, it does so at the single-cell level, and it capitalizes on powerful new techniques for examining gene function.